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| Product Name | APO-DIRECT TUNEL Assay Kit (FITC-dUTP Labeling for Flow Cytometry) |
| Catalog No. | CCAT-HMM-0060 |
| Description | A single-step TUNEL (Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling) assay kit for direct detection of DNA strand breaks in apoptotic cells by flow cytometry. The kit uses FITC-conjugated dUTP for direct fluorescent labeling of DNA breaks, eliminating secondary detection steps and reducing non-specific background. Optimized for quantitative measurement of apoptosis in suspension and adherent cell populations. |
| Intended Use | Quantitative detection of apoptotic cells by flow cytometry; DNA fragmentation analysis; evaluation of apoptosis-inducing agents; chemotherapy and radiotherapy response assessment; discrimination of apoptosis from necrosis. |
| Principle / Technology | Terminal deoxynucleotidyl transferase (TdT) catalyzes template-independent addition of FITC-conjugated dUTP to free 3'-OH termini of DNA strand breaks generated during apoptosis; fluorescent labeling intensity is proportional to the degree of DNA fragmentation |
| Detection Method | Flow cytometry; excitation 488 nm, emission 530/30 nm (FL1 or FITC channel); also compatible with fluorescence microscopy on cytospin preparations |
| Sample Type | Single cell suspension from cultured cells (adherent or suspension), dissociated tissue; requires 0.5–2 × 10^6 cells per sample; include positive and negative control cells |
| Sensitivity / LOD | Detects apoptotic populations as low as 1–2% of total cells; resolves early apoptotic cells (before plasma membrane damage) from late apoptotic/necrotic cells; linear response over 0–100% apoptosis |
| Specificity | Specific for free 3'-OH DNA ends generated during apoptosis; necrotic cells show lower labeling due to random DNA degradation; TdT enzyme is template-independent — does not require complementary strand |
| Reaction Conditions / Protocol | Fix cells in 1% paraformaldehyde (in PBS, pH 7.4) for 30–60 minutes on ice; wash; permeabilize with 70% ethanol at -20 °C for minimum 30 minutes (stable for up to 2 weeks); wash twice with wash buffer; resuspend in 50 µL DNA labeling solution (TdT enzyme + FITC-dUTP + reaction buffer); incubate 60 minutes at 37 °C; rinse twice with rinse buffer; resuspend in PI/RNase solution for counterstaining (optional); analyze by flow cytometry |
| Components / Formulation | TdT enzyme (30 U/µL), FITC-dUTP nucleotide mix, reaction buffer (5×), wash buffer (10×), rinse buffer (10×), positive control cells (lyophilized, fixed apoptotic cells), negative control cells (lyophilized, fixed non-apoptotic cells) |
| Storage Conditions | Store at -20 °C; protect FITC-dUTP from light; TdT enzyme is stable at -20 °C |
| Shelf Life | 12 months from manufacture date at -20 °C |
| Package Specifications | 25 tests, 50 tests, 100 tests |
| Product Form | Lyophilized control cells; liquid enzyme, nucleotide, and buffer reagents |
| Key Features | Direct FITC-dUTP labeling eliminates secondary antibody incubation; FITC provides bright signal compatible with standard flow cytometers; includes positive and negative control cells for assay validation; optimized fixation/permeabilization for minimal cell loss; validated on multiple cell types including lymphocytes, adherent lines, and primary cells |
| Purity | TdT enzyme specific activity >60,000 U/mg; FITC-dUTP incorporation efficiency >90% on positive control cells; no DNase or exonuclease contamination |
| Concentration | As specified per kit; sufficient for stated number of tests |
| Activity / Unit Definition | Apoptosis induction confirmed by positive control treatment producing ≥5-fold signal increase |
| Molecular Weight | Varies by dye and enzyme component |
| Source / Origin | Recombinant annexin proteins; synthetic fluorophore conjugates; chromogenic/fluorogenic peptide substrates |
| pH Range / Optimal pH | Binding/washing buffer pH 7.2–7.4 |
| Shipping Conditions | Dry ice -20 °C; do not allow to thaw during transit; FITC-dUTP must be protected from light |
| Expiration Date / Stability | 12 months at -20 °C; once thawed, TdT enzyme stable for 2 weeks at 4 °C; avoid repeated freeze-thaw cycles of enzyme; control cells stable for 6 months after reconstitution at 4 °C |
| Regulatory / Compliance | For laboratory and research use only; RUO; TdT is a research enzyme; FITC is a laboratory fluorophore; manufactured under ISO 9001 |
| Compatibility | Compatible with all flow cytometers with 488 nm excitation; use polypropylene or siliconized tubes to minimize cell adherence; avoid using PBS with Ca2+/Mg2+ during fixation which can activate endogenous nucleases |
| Recommended Buffer System | Annexin binding buffer with calcium; cell lysis buffer with protease inhibitors for caspase assays |
| Application Notes / Precautions | Always run a negative control (cells incubated with labeling solution minus TdT enzyme) to assess non-specific FITC-dUTP binding. Include positive control cells to verify enzyme activity. For adherent cells, trypsinize gently and wash thoroughly to remove trypsin which may degrade TdT. Analyze within 3 hours of staining for optimal FITC signal; if delayed, store cells at 4 °C in dark. |
| Batch-to-Batch Consistency | Positive control signal within ±20% of reference lot; staining pattern consistent |
For research use only, not for clinical use.
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